PROJECT SUMMARY Skin, the largest organ of the human body, is constantly exposed to solar ultraviolet radiation (UVR), a powerful environmental carcinogen. Unlike the skin of other mammals, human skin responds to UVR by increased pigmentation resulting from melanin production in epidermal melanocytes and its transfer to keratinocytes. Melanin protects the nuclear DNA of melanocytes and keratinocytes by directly absorbing UVR and neutralizing free radicals, thus preventing genetic damage and cancer formation. How does human skin detect and respond to UVR? UVR at the earth's surface consists of 95% long wavelength UVR (UVA) and 5% short wavelength UVR (UVB). Although the mechanism by which UVB causes DNA damage and results in delayed melanin synthesis more than a day after exposure is well characterized, the effects of UVA are poorly understood. High UVA doses cause oxidative damage, but no specific pathway for detecting physiological doses of UVA has been characterized. Our preliminary results describe a novel UVA-activated signaling pathway in melanocytes that is retinal-dependent and G-protein coupled. Activation of this pathway by physiological doses of UVA leads to the activation of transient receptor potential A1 (TRPA1) ion channels, a rapid increase in intracellular Ca2+, and increased melanin content on a rapid time scale (beginning about an hour after UVA exposure). The goal of this proposal is to characterize the molecular steps by which UVA causes a Ca2+ response (Aim 1) and an increase in cellular melanin concentration (Aim 2). Using live-cell fluorescence imaging, electrophysiology, and biochemistry, we will identify in Aim1 the G protein pathway activated by UVA, characterize the cellular messengers and determine the mechanisms that lead to a rapid and transient Ca2+ response. In Aim 2 we will test the hypothesis that UVA-mediated cellular depolarization is required for a sustained Ca2+ response and persistent PKC activation, which leads to early melanin production. In Aim 3 we will test the hypothesis that UVA and UVB signaling pathways, which are simultaneously activated by solar UVR, interact synergistically to regulate melanin production and pigmentation. We will use mice with humanized skin to investigate the interaction between the UVA and UVB activated pathways and characterize the molecular mechanisms mediating this interaction. The proposed studies will identify and characterize a novel UVA phototransduction pathway and determine its function in skin, and will significantly advance our understanding of melanocyte function and of the skin's response to solar UVR.